The Biowide project (2014-2018) was a project aiming at collecting biodiversity data from 130 terrestrial sampling sites across Denmark. Data was collected with both classical means (observation and trapping) and by eDNA metabarcoding (sequencing of amplified marker genes. Data was also collected on environmental variables. Biowide (Biodiversity in Width and Depth) took place in collaboration between Århus Universiity (main project holder) and the two major Danish natural history museums and the University of Copenhagen, and was supported by the Villum Foundation. During the first two years of the project, a host of professionals and volunteers collected data on the environment from the 130 sample plots – on everything from soil moisture, temperature, nutrients, plant biomass, trees, dead wood, stone and ant hills, expanses of water and flowers. The major effort has been collecting information on plants, mosses, lichens, invertebrates and fungi that live in the surfaces. With the help of volunteers, it was attempted to map as much as possible. As part of the Biowide project bulk soil samples were collected from each sampling site for eDNA metabarcoding (sequencing of amplified marker genes) with various genetic markers, for fungi, plants, insects, nematodes, etc. Sampling was stratified across the three gradients expected to have most importance for terrestrial biodiversity: fertility, humidity and disturbance. The plots represent one of the widest ecological samplings for eDNA to this date, as landscape types as diverse as sand dunes, meadows, bogs, agricultural land and also forest plantations and Danish natural forest are included. The project and methods are described in Brunbjerg et al. (2017) BIOWIDE eDNA Fungi dataset. As part of the Biowide project soil was collected for eDNA metabarcoding (sequencing of amplified marker genes) with various genetic markers, for fungi, plants, insects, nematodes, etc. This dataset represents data from the metabarcoding targeting soil fungi with primers (sub-)specific for fungi (gITS7 and ITS4), amplifying the ITS2 region.